The Ras/MAP kinase signaling pathway plays a central role in cell responses to growth factors as well as stress responses. It also plays a critical role in tumor progression. While it has been intensively studied over the last decade, many important questions remain unanswered. For example, while it is clear that Ras is activated at the plasma membrane, where and when Raf, MEK and ERK are activated is not known. A second issue concerns the architecture of the signaling pathway. While it has been suggested that the three-kinase cascade is designed for signal amplification or for "ultrasensitive signaling", these ideas are largely untested. Lastly, progress in signal transduction has seen the identification of scaffolds involved in organizing signaling pathways, but their effects on the magnitude of signaling responses and the quality of signal transduction is unknown. We have recently reported the phenotype of a knock-out mouse of what we believe to be the first scaffold in the mammalian MAP kinase signaling pathway. Here we propose to use this knockout mouse to explore the role of scaffolds in the Ras/MAP kinase signaling pathway. Because our understanding of these signaling pathways will rely on biophysical parameters that are not known, it will be important to measure and establish these parameters for each step of the pathway. Our first aim is to study the role of KSR in the interactions as well as on the kinetic enzymatic rate constants for each kinase in the cascade. This project mainly involves purification of proteins and biophysical measurements of affinity as well as enzymatic activity. Our second aim is to study the role of KSR in vivo. First, we focus on determining whether KSR effects the localization of MAP kinase signaling proteins in vivo as well as where MAP kinase components are activated. Secondly, we focus on effects of KSR on the kinetic of MAP kinase signaling at the single cell level. This includes measurements of the lag times between each step, the level of amplification as well as on the digital/analog quality of signaling. We hope that our studies will lead to a more comprehensive understanding of signaling pathways. Since these pathways are so critical to normal cell homeostasis, a better understanding of these pathways should also lead to a better understanding of what is happening when these pathways go awry in cancer, diabetes and autoimmune diseases.